The introduction of highly active antiretroviral therapy (HAART) has been successful in prolonging the lives of HIV/AIDS patients. However, long-term use of HAART, which is a combination therapy that almost invariably contains nucleoside HIV reverse transcriptase inhibitors (NRTIs), is associated with major toxicities including liver damage, neuoropathy, pancreatitis, myopathy, neuropathy, lactic acidemia and lipodystrophy, some of which could result in patient fatalities. The culprits for these toxicities are believed to be the NRTIs in HAART. These nucleoside drugs, including zidovudine (AZT), stavudine (d4T), didanosine (DDI), zalcitabine (DDC) and lamivudine (3TC) are believed to cause mitochondria! depletion to varying extents, leading to cell death and tissue toxicity. This mitochondrial depletion stems mainly from the inhibition of mitochondrial (mt) DNA polymerase gamma by the triphosphate metabolites of NRTIs. The entry of nucleosides into mitochondria and cells occurs through specialized membrane carrier proteins termed nucleoside transporters. Upon entry into mitochondria, nucleosides are sequentially phosphorylated by mitochondrial kinases such as the mitochondrial-specific thymidine kinase (TK-2) and others to yield the active triphosphate metabolites, which then inhibit mtDNA synthesis. It has recently been shown that mitochondria in mammalian cells express the equilibrative nucleoside transporter 1 (ENT1) in their membranes and that this expression enhances mitochondrial toxicity of antiviral nucleoside drugs. In light of this observation, we hypothesize that selective inhibition of mitochondrial nucleoside transporters can prevent entry of NRTIs into mitochondria of patients undergoing HAART, can be used as an approach to reduce the mitochondrial toxicity of these anti-HIV drugs. The following specific aims will be pursued in exploring this strategy for reducing NTRI mitochondrial toxicity. 1) Synthesize and characterize novel ester prodrugs of the nucleoside transporter inhibitor dipyridamole in terms of ENT transporter inhibition, cellular permeation and enzymatic ester hydrolysis. 2) Determine the ability of the appropriate dipyridamole ester prodrug to protect against mitochondrial toxicity of anti-HIV nucleosides. Methods will include synthetic and analytical chemistry, nucleoside transporter binding and nucleoside uptake assays, flow cytometry and real-time PCR. If successful, this research will increase our understanding of the role of nucleoside transporters in mitochondrial toxicity and could lead to a new strategy for reducing or preventing NRTI toxicities of long-term use of HAART in HIV/AIDS patients, and provide a novel pharmacological approach to addressing mitochondrial toxicity of nucleoside analog antiviral or anticancer agents, as well as furnish new knowledge on the effects of inhibiting mitochondrial nucleoside transport in situ.